DESCRIPTION (Applicant's Abstract): Dopamine reuptake at the plasma membrane by the dopamine transporter (DAT) is a major mechanism for terminating dopaminergic synaptic transmission. DAT and the related sodium- and chloride-coupled neurotransmitter transporters combine functional aspects of both G-protein-coupled-receptors and ion channels: namely binding sites for substrate, inhibitors, and ions, and a gated channel or transport pathway through which substrate and ions move. Binding of substrate, sodium and chloride mediates a conformational change which exposes the substrate and ions to the intracellular environment where they are released. Therefore, a water-accessible transport pathway must be formed among the membrane-spanning segments. This pathway should be accessible to hydrophilic reagents applied extracellularly. Although they may not be identical, the binding sites for substrate, ions and inhibitors, such as cocaine, likely lie, at least in part, within this transport pathway. We have developed an approach, the substituted-cysteine-accessibility method, to obtain information about the structure of binding sites and channels by systematically identifying the residues which line the site or channel. Our approach combines: site-directed mutagenesis to replace putative membrane-spanning segment residues, one at a time, with cysteine; heterologous expression of the mutant; and probing the aqueous surface accessibility of the engineered cysteine residue by its ability to react with small, charged, hydrophilic, lipophobic, sulfhydryl-specific reagents. The long-term goals of this project are to determine the structural bases of the transport of substrate by DAT and its inhibition by drugs such as cocaine. The specific aims are: l) To identify the amino acid residues forming the surface of the cocaine binding site, the dopamine binding site, and the transport pathway in DAT. 2) To determine the secondary structure of the membrane-spanning segments containing these residues. 3) To identify conformational changes of the membrane-spanning segments associated with transport. The approach outlined in this proposal will enable us to create a low resolution structural model of DAT, thereby laying a foundation for understanding, at the molecular level, the binding and transport of dopamine and its inhibition by cocaine. This approach might lead to a differentiation of the binding sites for cocaine and for dopamine and thereby facilitate the development of cocaine antagonists which do not inhibit dopamine transport. Furthermore, the approach will provide insights into structure-function relationships for other members of the neurotransmitter transporter family, such as the serotonin transporter and norepinephrine transporter, which are targets for a wide variety of antidepressant drugs.